Isolated hepatocytes maintained in culture have been used to study carbon tetrachloride (CCl4) induced hepatotoxicity. Hepatocytes maintained in cultured for 1 day retain the capacity to metabolize aminopyrine and to activate CCl4. Cell injury has been assessed by measuring leakage of cytoplasmic enzymes (e.g., lactic dehydrogenase: LDH), lifting of cells from the monolayer, morphological appearance and exclusion of trypan blue at different calcium concentrations and in the presence of the calcium ionophore A23187. No significant changes in covalent binding of CCl4 are observed under these culture conditions. Release of LDH with ionophore (1uM) or CCl4 (1.5 uM) alone is decreased by complexing calcium with EGTA (5uM). Injury due to ionophore and CCl4, in the presence of high calcium, is approximately additive, and is characterized by extensive "blebing" of the plasma membrane when these agents are used in combination. The results reported here and by other laboratories implicate calcium as a modulator of cell injury by CCl4.